Genetic modification techniques enable one to insert exogenous nucleotide sequences into an organism's genome. A number of methods have been described for the genetic modification of plants. All of these methods are based on introducing a foreign DNA into the plant cell, isolation of those cells containing the foreign DNA integrated into the genome, followed by subsequent regeneration of a whole plant. Unfortunately, such methods produce transformed cells that contain the introduced foreign DNA inserted randomly throughout the genome and often in multiple copies.
The random insertion of introduced DNA into the genome of host cells can be lethal if the foreign DNA happens to insert into, and thus mutate, a critically important native gene. In addition, even if a random insertion event does not impair the functioning of a host cell gene, the expression of an inserted foreign gene may be influenced by "position effects" caused by the surrounding genomic DNA. In some cases, the gene is inserted into sites where the position effects are strong enough to prevent the synthesis of an effective amount of product from the introduced gene. In other instances, overproduction of the gene product has deleterious effects on the cell.
Transgene expression is typically governed by the sequences, including promoters and enhancers, which are physically linked to the transgene. Currently, it is difficult to precisely modify the structure of transgenes once they have been introduced into plant cells. In many applications of transgene technology, it would be desirable to introduce the transgene in one form, and then be able to modify the transgene in a defined manner. By this means, transgenes could be activated or inactivated where the sequences that control transgene expression can be altered by either removing sequences present in the original transgene or by inserting additional sequences into the transgene.
Therefore, it is essential to gain more control over foreign DNA integration into the nuclear genome of plant cells to expedite the efficient production of transgenic plants with stable and reliable expression of transgenic traits. Relatively low frequency and randomness of foreign DNA integration make genetic transformation a labor-intensive and unpredictable procedure. Multi-copy, random integrations of transforming DNA molecules frequently lead to aberrant expression of foreign genes, affect expression of endogenous genes, and provide transgenic organisms with unstable transgenic traits. All plant transformation procedures currently in use take advantage of biochemical pathway(s) involving random, illegitimate recombination to integrate foreign DNA. Illegitimate recombinations constitute the intrinsic property of a conventional genetic transformation process. As such, desired DNA integration events cannot be separated, or preferably selected for, from among any excessive random integrations, unless a different mechanism governs the integration of productive events.
One approach for gene targeting, which is extensively pursued, involves the use of DNA homologous recombination for integration of foreign DNA into pre-selected genomic locations. The process involves both productive (homologous, targeted) and non-productive (illegitimate, random) integrations. Innovative strategies have already been proposed to reduce, or eliminate random integration of targeting vectors. They include the use of negative selection markers to eliminate random integrations by selection against actively expressed foreign genes, excisions of randomly integrated copies of foreign genes by the use of site-specific recombinations, or identification and application of specific inhibitors of non-homologous recombinations such as poly-(ADP-ribosylation) inhibitors.
The basic problem with current gene targeting procedures, however, is that the efficiency of homologous recombination in somatic cells of higher eukaryotes is extremely low being about 1,000-, 1,000,000-fold less frequent than illegitimate, random integrations. Taking into account that random integrations are barely considered satisfactory in the conventional genetic transformation procedures, routine gene targeting is presently not practical, at least in plant genetic transformation systems. Therefore, methods to control targeting and integration of foreign genes into the genome are needed.